Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation
Article first published online: 27 JUL 2007
Copyright © 2007 Wiley-Liss, Inc.
Microscopy Research and Technique
Volume 70, Issue 10, pages 880–885, October 2007
How to Cite
Hayek, A., Grichine, A., Huault, T., Ricard, C., Bolze, F., Van Der Sanden, B., Vial, J.-C., Mély, Y., Duperray, A., Baldeck, P. L. and Nicoud, J.-F. (2007), Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation. Microsc. Res. Tech., 70: 880–885. doi: 10.1002/jemt.20493
- Issue published online: 17 SEP 2007
- Article first published online: 27 JUL 2007
- Manuscript Accepted: 22 MAR 2007
- Manuscript Received: 11 JAN 2007
- University Louis Pasteur of Strasbourg
- two-photon dyes;
- cell microscopy;
- mouse brain imaging;
Because of the spreading of nonlinear microscopies in biology, there is a strong demand for specifically engineered probes in these applications. Herein, we report on the imaging properties in living cells and nude mice brains of recently developed water soluble blue fluorophores that show efficient diffusion through cell membranes and blood–brain barriers. They are characterized by two-photon absorption cross-sections of 100–150 Goeppert-Mayer range in the near IR and fluorescence efficiencies of up to 72% in water. They were found to stain homogeneously the cytoplasm of cultured living cells within minutes. Moreover, their diffusion times and fluorescence characteristics in the cytoplasm suggest a hydrophobic association with intracellular membranes. Their intracellular fluorescent decays were found to be almost mono-exponential, a very favorable feature for fluorescence lifetime imaging. Two photon images of living cells were obtained with a good signal to noise ratio using laser powers in the sub-milliwatt range. This allows continuous imaging without significant photobleaching for tens of minutes. In addition, these fluorophores allowed in vivo three-dimensional two-photon imaging of mice cortex vasculatures and extra vasculature structures, with no sign of toxicity. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.